Nucleic acids encoding ligands for HEK4 receptors

ABSTRACT

Polypeptides which bind to one or more EPH-like receptors, particularly the HEK4 receptor, are described. The polypeptides are designated HEK4 binding proteins. Nucleic acids encoding HEK4 binding proteins, and expression vectors, host cells and processes for the production of the polypeptides are also described. The polypeptides are useful for modulating the growth and/or differentiation of a variety of tissues, including those from liver, kidney, lung, skin, digestive tract and nervous system and may be used to regenerate damaged or depleted tissue and to treat cancer or nervous system disorders.

The invention relates to polypeptides which bind to one or more EPH-likereceptors. More particularly, the invention relates to polypeptideswhich bind to the HEK4 receptor, to nucleic acids encoding same and toexpression vectors and host cells for the production of thepolypeptides.

BACKGROUND OF THE INVENTION

The response of cells to their environment is often mediated by solubleprotein growth and differentiation factors. These factors exert theireffects by binding to and activating transmembrane receptors. Thisinteraction is the initial event in a cascade which culminates in abiological response by the cell. An important class of transmembranereceptors is the receptor protein tyrosine kinases (receptor PTKs,reviewed in van der Geer et al. Ann. Rev. Cell. Biol. 10, 251-337(1994). PTKs consist of an extracellular domain which interactsspecifically with the receptor's cognate ligand, a membrane spanningdomain, and an intracellular domain which harbors the tyrosine kinaseactivity. Receptor PTKs are activated by ligand-mediated dimerizationfollowed by autophosphorylation of tyrosine residues in the cytoplasmicdomain. The receptor PTK can then in turn phosphorylate substratemolecules in the signal transduction pathway, leading to a cellularresponse.

The family of receptor PTKs can be divided into a number of sub-familiesbased on the general structure of the extracellular domain and on aminoacid sequence relationships within the catalytic domain. Currently, thelargest known sub-family of receptor protein tyrosine kinases is theEPH-like receptors, consisting of at least 13 members. Members of thissub-family include the following: EPH (Hirai et al., Science 238,1717-1725 (1987)), ECK (Lindberg et al., Mol. Cell. Biol. 10, 6316-6324(1990)), Cek4, Cek5, Cek6, Cek7, Cek8, Cek9, Cek10 (Pasquale, CellRegulation 2, 523-534 (1991); Sajjadi et al., The New Biologist 3,769-778 (1991); Sajjadi and Pasquale Oncogene 8, 1807-1813 (1993)), Eek,Erk (Chan and Watt, Oncogene 6, 1057-1061 (1991)), Ehk1, Ehk2(Maisonpierre et al., oncogene 8, 3277-3288 (1993)), HEK (PCTApplication No. WO93/00425; Wicks et al., PNAS 89, 1611-1615 (1992)),HEK2 (Bohme et al., Oncogene 8, 2857-2862 (1993)), HEK5, HEK7, HEK8,HEK11 (U.S. Ser. No. 08/229,509) and HTK (Bennett et al. J. Biol. Chem.269, 14211-14218 (1994)).

Until recently, no ligands for any member of the EPH sub-family had beenidentified. A ligand for the Eck receptor was described in PCTApplication No. WO 94/11020 and Bartley et al. (Nature 368, 558-560(1994)) and identified earlier as B61, a polypeptide encoded by a cDNAof unknown function (Holzman et al., Mol. Cell Biol. 10, 5830-5838(1990)). Ligands for Elk and Ehk1 receptors have also been reported (PCTApplication No. WO094/11384; Davis et al., Science 266, 816-819 (1994)).Most recently, a polypeptide (ELF-1) identified from a mouse embryomidbrain and hindbrain cDNA library has been reported to be a ligand forMek4 and Sek (Cheng and Flanagan, Cell 79, 157-168 (1994).

Most attempts to purify soluble factors from complex biological fluidshave depended on cell-based bioassays of the response to stimulation bythe factor. These include increased cell growth or survival, increasedDNA synthesis, a chemotactic response, or some other downstreamconsequence of receptor activation. Receptor autophosphorylation hasalso been used as an assay to detect stimulation of the cell. We haverecently described a method for the isolation of ligands based on directdetection of receptor/ligand binding and the use of receptor affinitychromatography for purification (Bartley et al., supra). Here we reportthe application of this method to purify, sequence, and molecularlyclone one of a family of ligands corresponding to the EPH sub-family ofreceptor tyrosine kinases.

Although the EPH sub-family is the largest known sub-family of receptorPTKs, few ligands have been identified which bind to and activate an EPHsub-family receptor. It is therefore an objective to identify additionalligands for EPH sub-family receptor PTKs. These ligands will be usefulfor modulating responses of EPH sub-family receptor bearing cells.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides capable of binding to oneor more EPH-like receptor PTKs. More particularly, the inventionprovides polypeptides which bind to the HEK4 receptor, but may also bindto other members of the sub-family of EPH-like receptor PTKs. Thesepolypeptides are referred to as HEK4 binding proteins (HEK4 BPs). In oneembodiment, the polypeptide binds to and activates HEK4 and ECKreceptors. Also encompassed by the invention are nucleic acids encodingHEK4 BPs and nucleic acids which hybridize to HEK4 BP nucleic acids andencode polypeptides having at least one of the biological properties ofa HEK4 BP. Biologically active HEK4 BP fragments and analogs and nucleicacids encoding same as well as fusion proteins comprising HEK4 BP arealso encompassed by the invention.

Expression vectors and host cells for the production of biologicallyactive HEK4 BP and processes for the production of HEK4 BP using theexpression vectors and host cells are also within the scope of theinvention. Antibodies specifically binding HEK4 BP are also providedfor.

Polypeptides of the invention are useful for modulating (i.e.,increasing or decreasing) the growth and/or differentiation of EPHsub-family receptor-bearing cells, particularly cells expressing HEK4 orECK receptors. Based on levels of expression of HEK4, ECK, and HEK4 BPin a variety of tissues, it is expected that HEK4 BP will be useful formodulating the growth and/or differentiation, for example, liver,kidney, lung, skin or neural tissues. Administration of HEK4 BP tomammals is useful in the treatment of nervous system disorders and inthe regeneration of damaged or depleted tissues. HEK4 BP antagonists arealso useful for the treatment of cancers.

DESCRIPTION OF THE FIGURES

FIG. 1. BIAcore screening of conditioned media on HEK4-X surface.Concentrated samples of cell-conditioned media were screened on a HEK4-Xsurface as described in Example 2. The number of conditioned mediasamples giving a signal within each range of resonance units (RU) areshown in the histogram. Samples which bound more than 200 RU aresummarized in Table 1.

FIGS. 2A and 2B. Purification of HEK4 Binding Protein from A498conditioned media. C4 Reverse Phase HPLC column profile of HEK4 BP (A);SDS-PAGE analysis of pools of indicated peaks observed on the C4 column(B).

FIGS. 3A-C. Sequence of HEK-4 binding protein cDNA. The nucleic acidsequence of the human HEK-4 binding protein cDNA clone containing theentire coding sequence is shown along with the predicted amino acidsequence. The cDNA clone predicts a protein of between 213 and 228 aminoacids, depending on which of three potential start codons is utilized.The sequence is numbered so that the predicted mature N-terminal aminoacid is residue 1, with the putative signal peptide (underlined)extending from residues -19 to -1.

FIGS. 4A and 4B. Purification of recombinant HEK-4 binding protein. C4Reverse Phase HPLC column profile of recombinant HEK4 BP (A); SDS-PAGEanalysis of C4 fractions in the vicinity of the A₂₁₄ peak (B). Fractionsare identified by elution times from the C4 column.

FIG. 5. Expression of HEK-4 binding protein in human tissues. Theexpression of HEK-4 binding protein mRNA in human tissues was examinedby Northern blot analysis as described in Example 6. A blot containing 2μg of polyA+mRNA isolated from each of several tissues was purchasedfrom Clontech (Palo Alto, Calif.) and hybridized with a ³² P-labeledHEK-4 binding protein cDNA probe.

FIGS. 6A and 6B. Stimulation of tyrosine phosphorylation of EPH-likereceptors by membrane-bound HEK4 BP. CHO cells that express recombinantHEK4 receptor and endogenous ECK were treated with cells that weretransfected with an expression vector that contained the HEK4 BP cDNA orvector without cDNA. After lysis, HEK4 receptor (A) or ECK receptor (B)were immunoprecipitated. The immunoprecipitates were fractionated byPAGE, electroblotted, and probed with antiphosphotyrosine antibodies.

FIG. 7. Stimulation of tyrosine phosphorylation by soluble HEK4 BP.Cells were treated with conditioned media (CM) or recombinant HEK4 BP,with (+) or without (-) antibody clustering, and assayed for HEK4receptor activation. A) Twelve-fold concentrated media was compared to 2μg/ml HEK4 BP. (B) A dose response comparing clustered and unclusteredHEK4 BP.

FIG. 8. Relative affinity of HEK4 BP for HEK 4, ECK and HEK8 receptors.A competition assay for measuring binding of HEK4 BP to immobilized HEK4receptor in the presence of increasing concentrations of soluble HEK4,ECK and HEK8 receptors was performed as described in Example 8. The lineidentified as "IC 50" is drawn at an RU value corresponding to a HEK4 BPconcentration which is 50% of the control (no competitor) RU value.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to polypeptides capable of binding to oneor more EPH-like receptor PTKs and, more particularly, are capable ofbinding to a human homolog of an EPH-like receptor PTK. To date, eighthuman homologs of EPH-like receptors have been identified: EPH, ECK,HTK, HEK2, HEK4, HEK5, HEK7, HEK8 and HEK11. Characteristics of severalHEK receptors are disclosed in co-pending and commonly owned U.S. Ser.No. 08/229,509 herein incorporated by reference. The polypeptides of thepresent invention preferentially bind HEK4 receptor and are referred toherein as HEK4 binding proteins (HEK4 BP). The HEK4 receptor is aglycosylated 135 kDa protein tyrosine kinase previously identified asHEK receptor by Wilks et al. supra, and is the human homolog of the Cek4 and Mek 4 receptors identified in chicken and mouse, respectively.Polypeptides capable of binding HEK4 receptor may also activate thereceptor by inducing receptor autophosphorylation, an event whichinitiates transmission of a signal from the cell surface to the nucleus.Activation of HEK4 receptors leads to modulation of growth and/ordifferentiation of HEK4 receptor-bearing cells. HEK4 BP also binds toand activates other EPH-like receptors as described below.

A HEK4 binding protein has been identified and isolated from A498 cellline conditioned medium by procedures generally described in U.S. Ser.No. 08/145,616, relevant portions of which are herein incorporated byreference, and described in more detail in Example 2. Briefly, a geneencoding the extracellular domain of the HEK4 was constructed andexpressed as described in Example 1. The purified HEK4 extracellulardomain was immobilized on a BIAcore sensor chip and concentratedconditioned media from 102 different cell lines were screened forbinding to HEK4 receptor extracellular domains by surface plasmonresonance. This procedure identified conditioned medium from severalcell lines shown in Table 1 having one or more factors which interactedwith HEK4. The A498 cell line was chosen as a source for HEK4 ligand anda protein binding to HEK4 was purified as described in Example 2.Purified and isolated HEK4 binding protein from A498 cell-conditionedmedium has three major forms of molecular weights 21, 25 and 27 kD onnonreducing SDS-polyacrylamide gels. These forms represent glycosylationand C-terminal processing variants. HEK4 BP has the amino acid sequencesas shown in Table 2 for the peptides generated by cyanogen bromide ortrypsin cleavage.

cDNA clones of HEK4 binding protein were obtained from a human placentacDNA library as described in Example 3. The sequence of human HEK4binding protein cDNA is shown in FIGS. 3A-C. Based upon cDNA sequencingand carboxy-terminal peptide mapping of the A498 cell-derived protein, amajor secreted form of HEK4 binding protein had an amino terminal serineresidue as shown in FIGS. 3A-C and a carboxy terminal proline residue atposition 179. An alternate secreted form having a carboxy terminalalanine residue at position 177 was also detected. Alternative forms ofHEK4 BP, including membrane-bound forms, may also be synthesized.

Recombinant HEK4 binding protein was expressed in CHO cells transfectedwith cDNA encoding HEK4 BP as shown in FIGS. 3A-C. Soluble HEK4 BP waspurified as described in Example 4 and shown to bind HEK4 receptor byBIAcore analysis. Purified soluble HEK4 BP activated HEK4 receptor andthis activation was enhanced by antibody clustering of the ligand (FIGS.7A, 7B). Activation of HEK4 receptor was also observed upon contact ofCHO cells expressing HEK4 BP with CHO cells expressing HEK4 receptor(FIGS. 6A, 6B). Therefore, recombinant HEK4 binding protein binds andactivates EPH-like receptor PTKs.

The invention provides for a purified and isolated polypeptide, termedHEK4 binding protein, capable of binding at least one EPH-like receptor.In one embodiment, the polypeptide is capable of binding the HEK4receptor. HEK4 binding protein is mammalian and is preferably human.Purified HEK4 binding protein is substantially free of other humanproteins and has a molecular weight of about 21 to 27 kD on nonreducingSDS-PAGE. HEK4 BP has at least about 70% homology to the amino acidsequence as shown in FIGS. 3A-C (SEQ ID NO: 1) and is capable of bindingat least one EPH-like receptor. Preferably, HEK4 BP has the amino acidsequence as shown in FIGS. 3A-C (SEQ ID NO: 1). Binding of an EPH-likereceptor by HEK4 BP may or may not result in receptor activation.EPH-like receptor binding and activation may be effected by eithersoluble or membrane-bound form of HEK4 BP, or by both forms. It isfurther understood that receptor binding and activation by HEK4 BP isnot restricted to HEK4 receptor, but that HEK4 BP may also bind to andactivate other EPH-like receptor family members. As described in Example7, HEK4 binding protein has been shown to activate both HEK4 and ECKreceptors.

HEK4 binding proteins of the invention are preferably characterized bybeing the product of procaryotic or eucaryotic expression of anexogenous DNA sequence, i.e., HEK4 binding protein is a recombinantprotein. Exogenous DNA is DNA which encodes HEK4 binding protein andincludes cDNA, genomic DNA and synthetic (manufactured) DNA. HEK4binding protein may be expressed in bacterial, yeast, plant, insect ormammalian cells in culture or in transgenic animals using DNA expressionvectors appropriate for the given host cell. Expression of recombinantHEK4 binding protein in CHO cells is described in Example 3A, 3B, 3C ofthe specification.

Also provided is HEK4 BP in dimeric or higher order oligomeric stateswherein multimeric HEK4 BP is capable of binding and/or activatingEPH-like receptors. Soluble HEK4 BP multimers are selected from thegroup consisting of HEK4 BP/immunoglobulin chimeras, HEK4 BP clusteredby treatment with anti-HEK4 BP antibodies, and covalently andnoncovalently attached HEK4 BP monomers. Clustered HEK4 BP is describedin Example 7B and HEK4 BP chimeras are constructed using standardrecombinant DNA techniques. Covalently and noncovalently attached HEK4BP monomers are produced using protein crosslinking reagents andprocedures readily available to one skilled in the art.

The polypeptides of the present invention include biologically activefragments and analogs of HEK4 BP. HEK4 BP fragments encompass amino acidsequences having truncations of one or more amino acids from thesequence shown in FIGS. 3A, 3B, 3C or in SEQ ID NO: 1, wherein thetruncation may originate from the amino terminus, carboxy terminus, orfrom the interior of the protein. Analogs of the invention involve aninsertion or a substitution of one or more amino acids within thesequence as shown in FIGS. 3A-C or SEQ ID NO: 1. Fragments and analogswill have at least one biological property of HEK4 binding protein,typically the ability to bind at least one EPH-like receptor.

Also encompassed by the invention are chimeric polypeptides comprisingHEK4 BP amino acid sequences fused to heterologous amino acid sequences.Said heterologous sequences encompass those which, when formed into achimera with HEK4 BP, retain one or more biological or immunologicalproperties of HEK4 BP. In one embodiment, a HEK4 BP/immunoglobulinchimeric protein is encompassed wherein chimeric molecules may aggregateto multimeric forms of HEK4 BP for receptor binding and activation. Oneexample is a chimera of HEK4 BP and the Fc region of IgG.

Also provided by the invention is an isolated nucleic acid encoding HEK4binding protein. The nucleic acid is selected from the group consistingof:

a) the nucleic acid as shown in FIGS. 3A-C (SEQ ID NO: 1);

b) nucleic acids which hybridize under conditions of 6×SSC and 65° C.with the coding regions as shown in FIGS. 3A, 3B, 3C (SEQ ID NO: 1);

c) nucleic acids which are degenerate to the nucleic acids of (a) and(b). The nucleic acids may be cDNA, genomic DNA or synthetic(manufactured) DNA. It is understood that the hybridization conditionsspecified herein allow one skilled in the art to estimate the extent ofmismatch between a given nucleic acid and a nucleic acid comprising thecoding region as shown in FIGS. 3A-C (SEQ ID NO: 1) and that suchconditions may be varied by changing salt, temperature and/or length ofincubation or adding organic solvent at either the washing orhybridization steps and still allow one to obtain an equivalent level ofmismatch during hybridization. Therefore, it is envisioned that thenucleic acids of the invention include those which hybridize with thecoding regions in FIGS. 3A-C under conditions equivalent to those of6×SSC and 65° C. Nucleic acid sequences encoding HEK 4 binding proteinmay have an amino terminal leader sequence and a carboxy terminalmembrane anchor sequence or alternatively, may have one or bothsequences removed. The encoded polypeptides will have at least one ofthe biological properties of HEK4 BP.

The nucleic acids of the invention will be operatively linked withnucleic acid sequences so as to express HEK4 binding protein. Sequencesrequired for expression are known to those skilled in the art andinclude promoters and enhancer sequences for initiation oftranscription, transcription termination sites, ribosome binding sites,and sequences directing polypeptide secretion. A general description ofnucleic acid sequences which serve to direct expression of exogenousgenes is found in Methods in Enzymology v. 185, D. V. Goeddel, ed.Academic Press, Inc. New York (1990). Sequences directing expression ofHEK4 binding protein may be homologous or heterologous. A variety ofexpression vectors may be used to express HEK4 binding protein inprocaryotic or eucaryotic cells in culture. One such vector is pDSRαdescribed in PCT Application No. WO90/14363 which was used to expressHEK4 BP in CHO cells (see Example 3). In addition, vectors fortissue-specific expression of HEK4 binding protein in transgenic animalsand viral-based gene transfer vectors for expression of HEK4 bindingprotein in human cells in vivo are also available. The nucleic acidcoding regions of HEK4 binding protein may be modified by substitutionof preferred codons for optimal expression in an appropriate host cellusing procedures available to the skilled worker.

Plasmid pDSRα containing the nucleic acid sequence encoding HEK4 BP asshown in FIGS. 3A, 3B, 3C has been deposited with the American TypeCulture Collection, Rockville, Md. on Jan. 20, 1995, under ATCCAccession No. 97028.

A host cell transformed or transfected with nucleic acids encoding HEK4binding protein are also encompassed by the invention. Any host cellwhich produces a polypeptide having at least one of the biologicalproperties of a HEK4 BP may be used. Specific examples includebacterial, yeast, plant, insect or mammalian cells. In addition, HEK4binding protein may be produced in transgenic animals. Transformed ortransfected host cells and transgenic animals are obtained usingmaterials and methods that are routinely available to one skilled in theart. Host cells may contain nucleic acid sequences having thefull-length gene for HEK4 binding protein including a leader sequenceand a C-terminal membrane anchor sequence (as shown in FIGS. 3A-C) or,alternatively, may contain nucleic acid sequences lacking one or both ofthe leader sequence and the C-terminal membrane anchor sequence. Inaddition, nucleic acid fragments, variants and analogs which encode apolypeptide capable of binding HEK4 receptor may also be resident inhost expression systems. Polypeptides of the invention are produced bygrowing transformed or transfused host cells under suitable nutrientconditions to express HEK4 BP and isolating the results at polypeptides.

Antibodies specifically binding HEK4 binding proteins of the inventionare also encompassed. The antibodies can be produced by immunizationwith full-length (unprocessed) HEK4 binding protein or its mature formsor a fragment thereof. Antibodies may be polyclonal or monoclonal andmay be human or murine-derived. Antibodies of the invention may also berecombinant, such as chimeric antibodies having the murine constantregions on the light and heavy chains replaced by human constant regionsequences; or complementary determining region (CDR)-grafted antibodieswherein only the CDR is of murine origin and the remainder of theantibody chain has been replaced by human sequences.

The invention also provides for a pharmaceutical composition comprisinga therapeutically effective amount of HEK4 binding protein and apharmaceutically acceptable adjunct. Examples of pharmaceuticallyacceptable adjuncts include diluents (Tris, acetate or phosphatebuffers), carriers (human serum albumin), solubilizers (Tween,polysorbate), preservatives (thimerosol, benzyl alcohol) andanti-oxidants (ascorbic acid). A more extensive survey of componentstypically found in pharmaceutical compositions appears in Remington'sPharmaceutical Sciences 18th ed. A. R. Gennaro, ed. Mack, Easton, Pa.(1990). As used herein, the term "therapeutically effective amount"refers to that amount of HEK4 binding protein which provides atherapeutic effect for a given condition and administration regimen.Said therapeutically effective amount may vary from 0.01 μg/kg bodyweight to 10 mg/kg body weight and may be determined by one skilled inthe art.

HEK4 binding protein may be administered by injection, eithersubcutaneous, intravenous or intramuscular, or by oral or nasaladministration. The route of administration to be chosen will dependupon several variables, including the nature and severity of thecondition being treated and the pharmacokinetic properties of the HEK4binding protein preparation. HEK4 binding protein may be formulated fordelivery in a particular fashion, e.g., it may be modified with watersoluble polymers, such as polytheylene glycol to improve properties fornasal delivery or to improve serum half-life after injection; or it maybe incorporated into particulate preparations of polymeric compounds(e.g., liposomes) for controlled delivery over an extended period oftime.

The expression of HEK4 receptor and HEK4 BP in various tissues isreported in Examples 6A and 6B, respectively. HEK4 receptor mRNA wasmost abundant in human placenta and was also detected in heart, brain,lung, liver, muscle, kidney tissues. HEK4 BP mRNA was most abundant inhuman adult brain, kidney and placenta, and was detected at lower levelsin heart, lung, liver, spleen, prostate, testis, ovary, small intestine,muscle, pancreas and colon. These patterns of expression suggest thatactivation of HEK4 receptor by HEK4 BP modulates the growth and/ordifferentiation of a variety of target cells, particularly those in thebrain, heart, lung, liver, muscle and pancreas where expression of bothreceptor and ligand are detected. In addition, Wicks et al., supra hasreported HEK4 receptor mRNA in pre-B and T cell lines, suggesting a rolefor HEK4 BP in hematopoiesis.

As described in Example 7, HEK4 BP also activates ECK receptor in acell-cell autophosphorylation assay. Eck receptor mRNA is most abundantin adult rat lung, small intestine, kidney, ovary and skin with lowerlevels detected in brain, spleen and submaxillary gland (Lindberg andHunter, supra). Recently, it has been shown that Eck is expressed in thenervous system of the early mouse embryo (Becker et al. Mech. Dev. 47,3-17 (1994); Ganju et al. Oncogene 9, 1613-1624 (1994)). Theseobservations suggest that activation of ECK receptor by HEK4 BP maymodulate the growth and/or differentiation of cells expressing ECK, suchas those in the lung, intestine, kidney, skin and nervous system.

Therefore, HEK4 BP is useful in modulating (i.e., increasing ordecreasing) the extent of growth and/or differentiation of target cellsin various tissues. The target cells will have at least one receptorwhich is activated by HEK4 BP wherein the receptor is preferably amember of the EPH sub-family of receptor PTKs. Potential therapeuticuses for HEK4 BP are described below.

One aspect of the invention is the use of HEK4 BP to modulate cellgrowth and differentiation in the nervous system. HEK4 BP may be used tomaintain or restore cellular function in the nervous system of a mammalwhich has been decreased or eliminated by disease or injury or is atrisk of being decreased or eliminated by disease or injury. Target cellsinclude neurons and glial cells. Conditions that may be treated by HEK4BP include central nervous system disorders such as Alzheimer's disease,Parkinson's disease, multiple sclerosis, stroke and Huntington's diseaseand peripheral nervous system disorders such as amyotrophic lateralsclerosis (ALS) and peripheral neuropathies. Physical injuries to thespinal cord and to peripheral neurons may also be treated with HEK4 BP.

Another aspect of the invention is the modulation by HEK4 BP of growthand differentiation of digestive tract (including large and smallintestine), liver, lung, pancreas, muscle and hematopoietic tissues.This activity of HEK4 BP may be particularly useful in regeneration oftissue in these and other sources which has been damaged or depleted bydisease or injury.

It has been observed that the sub-family of EPH-like receptors and theircorresponding ligands are highly expressed in some carcinoma cell lines(see for example, PCT Application No. WO94/11020 for expression of ECKreceptor and ECK binding protein in human carcinoma cell lines). Thusanother aspect of the invention is the treatment of cancers using HEK4BP antagonists to block cell proliferation. Such cancers are likely tobe associated with organs which express HEK4 receptors and/or Eckreceptors. HEK4 BP antagonists may be any compound which blocks thebiological activity of HEK4 BP and may include, but are not limited to,the following: antibodies which bind to either HEK4 BP or to an EPHsub-family receptor which is activated by HEK4 BP such that areceptor/ligand interaction is prevented; HEK4 BP which binds to, butdoes not activate and EPH-like receptor; and soluble EPH-like receptorswhich bind to HEK4 BP. It is envisioned that small molecule mimetics ofthe above described antagonists are also encompassed by the invention.

In addition to in vivo applications, HEK4 BP may also be used ex vivo toamplify cell populations prior to transplantation. It is envisioned thatHEK4 BP may promote growth in culture of cells from the digestive tract,liver, lung, bone marrow, kidney, or central and peripheral nervoussystems (and glial cells) neurons such that the amplified population canbe introduced back into a patient in need of such therapy. Suchso-called "cell therapy" is useful in replenishing cells after damage ordepletion and may be appropriate under conditions where systemicadministration of HEK4 BP is not preferred.

HEK4 BP may be used alone or in combination with other therapeuticagents for the treatment of cancer, neurological disorders, disorders ofthe digestive tract, liver, or lung, and for the ex vivo expansion ofcell populations. HEK4 BP may be used in conjunction with otherchemotherapeutic drugs or with radiation therapy for the treatment ofcancer, or with other neurotrophic factors such as brain derivedneutrophic factor (BDNF), ciliary neurotrophic factor (CNTF),neurotrophin-3 (NT-3), nerve growth factor (NGF), or glial derivedneurotrophic factor (GDNF) for neurological disorders; or with tissuegrowth factors such as platelet derived growth factor (PDGF), fibroblastgrowth factor (FGF), epidermal growth factor (EGF), hepatocyte growthfactor (HGF) or keratinocyte growth factor (KGF) for restoration ofdamaged or depleted tissues.

Isolated nucleic acids of the present invention are useful reagents forthe detection and quantitation of DNA and/or RNA coding for HEK4 BP bystandard hybridization procedures such as those described in Sambrook etal. Molecular Cloning, A Laboratory Manual, 2d ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). These reagents allowone to determine the potential of various cell types to express HEK4 BPand related polypeptides and are also useful for detecting abnormalitiesin genes encoding HEK4 BP or in sequences controlling the expression ofHEK4 BP. Nucleic acids of the invention are also useful for controllingexpression levels of HEK4 BP. So-called "anti-sense" nucleic acidshybridize to DNA and/or RNA strands encoding HEK4 BP in a manner thatblocks transcription or translation of HEK4 BP nucleic acid sequences.Introduction of HEK4 BP anti-sense nucleic acids into cellsoverexpressing HEK4 BP is appropriate when such overexpression leads toundesirable physiological effects, such as excessive cell proliferation.

Antibodies which specifically bind HEK4 BP are useful reagents for thedetection and quantitation of HEK4 BP in biological samples usingimmunoassays (Western blots, RIAs, ELISAs) that are conventional to theart. The presence of HEK4 BP may be indicative of cell proliferation orthe potential for cell proliferation, and elevated levels may signalabnormal cell growth typically associated with cancer. In addition,antibodies of the invention may also be useful therapeutic reagents thatact as agonists or antagonists of HEK4 BP activity. Antibodies may bindto HEK4 BP in a manner that directly or indirectly blocks HEK4 BPbinding an EPH receptor (either HEK4 or ECK). Alternatively, antibodiesmay bind to HEK4 BP in a manner which promote receptor binding andactivation by, for example, "clustering" HEK4 BP into a dimeric orhigher multimeric forms to allow more efficient binding and activationof receptor. Antibodies can be monoclonal, polyclonal, or recombinant.

The following examples are offered to more fully illustrate theinvention, but are not construed as limiting the scope thereof.

EXAMPLE 1 Production of HEK4 Receptor Extracellular Doman (HEK4-X)

A cDNA clone coding for the HEK4 receptor protein tyrosine kinase wasisolated from a human fetal brain cDNA library (Stratagene, La Jolla,Calif.) as described in co-pending and commonly owned U.S. Ser. No.08/229,509. The sequence of this clone was identical to that in FIGS. 1of Wicks et al., supra with the following exceptions. Wicks reported thesequence TTA at nucleotides 1618-1620 whereas the HEK4 receptor cloneisolated as described here had the sequence TTC at these positions.However, Wicks et al.'s predicted protein sequence specifies aphenylalanine residue in this position, which is inconsistent with an"A" at nucleotide 1620 (TTA codes for leucine while TTC codes forphenylalanine). Also, nucleotides 1529 through 1531 of the Wicks et al.sequence are absent from the sequence obtained here. This change doesnot affect the translational reading frame, but does eliminate thepredicted glutamine residue at position 478 of the Wicks sequence. Theeffect of these differences on the biological activity of the receptoror the ability to bind ligand is unknown.

The HEK4 receptor cDNA clone was used as a template in a polymerasechain reaction (PCR) designed to amplify a DNA fragment coding for theligand binding domain of the HEK4 receptor. The primers used were:

433-26) 5' GGATCTAGAGCACCAGCAACATGGATTGT 3' (SEQ ID NO: 3)

409-10) 5' TCGGTCTAGATCATTATTGGCTACTTTCACCAGAGAT 3' (SEQ ID NO: 4)

These primers produce a fragment 1656 nucleotides in length that codesfor a protein of 540 amino acids. The predicted protein consists of theentire extracellular domain of the HEK4 receptor from the amino terminusup to but not including the transmembrane region. The 1656 nucleotidefragment was digested with the restriction endonuclease XbaI and ligatedinto the expression vector pDSRα which had been digested with the sameenzyme. The resulting expression plasmid was introduced into CHO cellsby calcium phosphate mediated transfection (Cellphect, Pharmacia,Piscataway, N.J.). Individual colonies were selected based upon theexpression of the dihydrofolate reductase (DHFR) gene which resides onthe expression plasmid. Expression of the HEK4 gene was monitored by RNAsolution hybridization (Hunt et al. Exp. Hematol. 19, 779-784 (1991))and/or by Western blotting with antibodies directed against amino acids22-148 of the HEK4 extracellular domain.

HEK4 expression was enhanced by growth of the selected clones in 100 nMmethotrexate. One of the pDSRα/HEK4-X clones was chosen for large scaleproduction. Twenty-four roller bottles were seeded at a density ofapproximately 2×10⁷ cells/bottle in 200 ml each of Dulbecco's MinimalEssential Media (DMEM) supplemented with non-essential amino acids (1×NEAA, Gibco), 100 nM methotrexate, 1× penicillin/streptomycin/glutamine(1× PSG, Gibco) and 10% fetal bovine serum. Cells reached confluence in3-4 days at which time the media was changed to DMEM/NEAA/PSG lackingserum. Cell-conditioned media was harvested after seven days,concentrated, and diafiltered against 10 mM Tris-HCl, pH 8.5. Theconcentrated media was loaded onto a Q-sepharose FF (Pharmacia) ionexchange column and bound material was eluted with a linear gradient of0 to 0.5 M NaCl in 10 mM Tris-HC1, pH 8.5. Fractions were analyzed bySDS-PAGE and western blotting using a rabbit polyclonal antibodydirected against residues 22-148 of the HEK4 external domain. Fractionscontaining HEK4-X protein were pooled, concentrated and loaded onto anS-200 (Sephaoryl S-200, Pharmacia) column. Fractions from this columnwere analyzed as before and those containing HEK4-X were pooled.

EXAMPLE 2 A. Purification of HEK4-X Binding Activity

We have previously described the use of the BIAcore™ instrument(Pharmacia Biosensor, Piscataway, N.J.) for the detection of receptorbinding activity in concentrated cell-conditioned media (Bartley et al.,supra). We used a similar strategy to screen for HEK4 receptor bindingactivity as described below.

The surface of a BIAcore sensor chip was activated by injection of 0.2 M1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl and 0.05 MN-hydroxysuccinimide at a flow rate of 5 ul/min. Purified HEK4-X at aconcentration of 250 μg/ml was applied to the activated surface in two50 ul injections at the same flow rate. Unreacted binding sites wereblocked by injection of 1M ethanolamine, pH 8.5. The surface was washedovernight in 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20, pH7.4 until the baseline was stable. Typically, immobilization resulted in6000-8000 resonance units (RU) of HEK4-X bound to the sensor chip.

Conditioned media samples were collected from 108 cell lines growneither without fetal bovine serum (FBS) or in the presence of 0.5% FBS.Conditioned media produced under serum-free conditions was adjusted to0.5% FBS before further processing. The media was filtered, concentrated25-fold, and stored in aliquots at -80° C. 30 ul samples of each mediumwere injected onto the HEK4-X surface at a flow rate of 5 μl/min and thebinding response measured 20 seconds after the conclusion of eachinjection. Between samples, the surface was regenerated by 10-15 μlinjections of 25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10.4.Concentrated conditioned media samples displaying binding of 200resonance units or more are listed in Table 1.

                  TABLE 1                                                         ______________________________________                                                                   Binding                                                                        Cell Line Description (resonance units)           ______________________________________                                        HCT116    human colon carcinoma                                                                          911                                                  M-14 human melanoma 337                                                       LS174T human colon adenocarcinoma 316                                         A498 human kidney carcinoma 274                                               A172 human glioblastoma 269                                                   PK(15)1 porcine kidney 234                                                    JEG-1 human choriocarcinoma 220                                               Y-79 human retinoblastoma 216                                                 HT 1080 human fibrosarcoma 200                                              ______________________________________                                    

The five conditioned media displaying the most binding were selected forfurther investigation. When soluble dextran was added to samples beforeinjection to reduce non-specific binding, the signals from HCT116 andLS174T conditioned media were greatly reduced. A172 cells proveddifficult to grow and were unsuitable for the large scale production ofconditioned media. Based on these experiments and on pilot scalereceptor affinity chromatography, the A498 cell line (ATCC No. HTB 44)was chosen as the best source of conditioned media for purification of aHEK4 binding protein.

A HEK4 receptor affinity column was prepared by immobilization of HEK4-Xon CNBr-activated Sepharose 4B (Pharmacia). Purified HEK4-X was dialyzedagainst 0.1 M NaHCO₃, 0.5 M NaCl, pH 8.3 and brought to a finalconcentration of 2 mg/mL. Immobilization of HEK4-X was done at a liganddensity of 1 mg/mL according to the method of Kenny et al. (New ProteinTechniques, J. M. Walker, ed. The Humana Press, Clifton, N.J. 1988).Forty liters of A498 conditioned media produced in 0.5% serum-containingmedia was concentrated 40-fold, diafiltered against PBS and 0.02% NaN₃,and loaded onto the HEK4-CNBr sepharose column. The column was washedwith PBS and bound material was eluted with 50 mM sodium acetate, 0.5 Msodium chloride, pH 4.0. Fractions were collected in 1 mM CHAPS andloaded directly on polyacrylamide gels. Gels were either stained withsilver for analysis or blotted onto PVDF membranes (Problot, AppliedBiosystems, Foster City, Calif.) in preparation for N-terminal aminoacid sequencing (Fausset & Lu, 1991).

The pH 4.0 elution fractions from the HEK4-CNBr Sepharose columncontained three major protein species with molecular weights of 21, 25and 27 kD which were not apparent in the load or wash fractions. Thefractions containing these 3 proteins were pooled, concentrated in thepresence of CHAPS and applied to a Vydac C4 reverse phase HPLC column(4.6×150 mm). The column was eluted with a gradient of acetonitrile(26-35%) in 0.1% trifluoroacetic acid. Fractions were collected, volumereduced under vacuum, and analyzed for HEK4 binding protein. The threemajor peaks detected by absorbance at 214 nm were pooled and analyzed bySDS-PAGE (see FIGS. 2A, 2B). Further purification of the three isoformsof HEK4 BP was achieved by reapplying the proteins to the same C4column.

B. Sequencing of Peptides

A sample from the initial purification of A498 cell-conditioned mediawas submitted for protein sequencing. The sample was analyzed onSDS-PAGE and blotted onto PVDF membrane. The gel band identified as theHEK4 binding protein was excised and analyzed for five cycles on anApplied Biosystems 477A protein sequencer. This yielded no sequence,indicating that the protein was N-terminally blocked. The sample wasthen treated with cyanogen bromide and reapplied to the sequencer. Atentative sequence was obtained from the cleaved sample by assuming thehighest yield at each cycle to belong to the same peptide. Even giventhis assumption, recoveries were so small as to render the sequenceunreliable after cycle 10. The sequence obtained in this manner is shownas peptide #1 in Table 2.

                  TABLE 2                                                         ______________________________________                                        Peptide No.                                                                            Amino acid sequence                                                  ______________________________________                                        1        Val-Asn-Phe-Asp-Gly-Tyr-Ser-Ala-Arg-                                     Asp (SEQ ID NO: 5)                                                           - 2 Val-Phe-Asp-Val-Asn-Phe-Lys-Val-Glu-X-                                     Ser-Leu-Glu-Pro-Ala-Asp (SEQ ID NO: 6)                                       - 3 Ala-Val-Ala-Asp-Arg-Tyr-Ala-Val-Tyr-                                       Trp-Asn-Ser-Ser-Asn-Pro-Arg-Phe-Gln-                                          Arg-Gly-Asp-Tyr-His-Ile-Ile-Val-X-Ile-                                        Asn-X-Tyr (SEQ ID NO: 7)                                                  ______________________________________                                    

Subsequent analysis of samples cleaved by cyanogen bromide, thenseparated by SDS-PAGE indicated that position 9 of peptide #1 was acysteine residue and position 6 of peptide #2 was aspartic acid.Position 25 of peptide #3 was subsequently found by DNA sequencing to beaspartic acid. The sequence data shown in peptides #2 and #3 in Table 2was obtained by analysis of tryptic digests of the protein followed byseparation of the resulting peptides on a microbore C4 column. Theseexperiments were done with larger amounts of starting material andtherefore yielded more reliable sequence and allowed 20-30 cyclesequencing runs. Comparison of the peptide sequences in Table 2 with B61suggests that they represent fragments of a related protein. Wetherefore conclude that the HEK4 binding protein is another ligand forthe human EPH-like kinase sub-family.

EXAMPLE 3 A. Cloning and Sequencing of cDNA Encoding HEK4 BindingProtein

The amino acid sequences obtained from HEK4 BP peptides as shown inTable 2 were used to design oligonucleotide primers. Primers 702-3 and633-11 were used in a PCR reaction with random primed A498 cDNA as atemplate.

702-3) 5' GAYMGNTAYGCNGTNTAYTGG 3' (SEQ ID NO:8)

633-11) 5' RTANCCRTCRAARTTNACCAT 3' (SEQ ID NO:9) The 175 base pairfragment amplified by these primers was sequenced and found to beclosely related to B61. This fragment was then radiolabeled with ³² P byrandom priming and used as a probe to screen a cDNA library for clonescontaining the full length HEK4 BP cDNA. An oligo-dT primed humanplacental cDNA library purchased from Stratagene (La Jolla, Calif.) wasplated at a density of 30,000 plaques/150 mm plate. Replicas of theplaques arrayed on the plates were made on GeneScreen™ hybridizationtransfer membranes (New England Nuclear, Boston, Mass.) as directed bythe manufacturer. Two replica filters were made for each plate. Filterswere pre-hybridized in 6× SSC, 1× Denhardts buffer, 50 ug/ml salmontestis DNA, 1% SDS at 65° C. for 4 hours followed by hybridization withthe ³² P-labeled probe for 12 hours under the same conditions. Followinghybridization, filters were washed two times, 1 hour each, in 0.2× SSC,0.5% SDS at 65° C. and exposed to Kodak XAR film overnight with anintensifying screen. Comparison of the two filters made from each plateshowed that five plaques were positive on both replicas. The phagearound each positive plaque were removed, resuspended in buffer, andreplated at a lower density to produce well-separated plaques forsecondary screening. Individual plaques which were positive uponrescreening (using the same method as the primary screen) were picked.The inserts from these phage were transferred into the pBluescriptplasmid by in vivo excision as described by the manufacturer(Stratagene). Three of the five inserts were identical and contained theentire coding region of HEK4 BP while the other two representedoverlapping clones. A consensus sequence was assembled using data fromthe three inserts containing the entire coding region and is shown inFIGS. 3A-C.

The HEK4 BP cDNA sequence predicts a protein of between 213 and 228amino acids, depending on which of three possible initiator codons isutilized. Based upon rules for translation of vertebrate mRNAs (KozakCell 44, 283-292 (1986)), the third in-frame ATG is an unlikelyinitiator, while the first ATG, being the farthest upstream is the mostlikely to be the principal initiation codon. As for B61, HEK4 BP hashydrophobic amino acids on both the amino and carboxy termini. Theseprobably function as a secretion signal sequence and a membrane anchor,respectively. Like B61, HEK4 BP apparently has both soluble and membranebound forms. Although we were not able to obtain N-terminal proteinsequence data, we would predict cleavage of the signal peptide to yielda mature protein with serine at position 1 (FIGS. 3A, 3B, 3C). Based onpeptide mapping and mass spectrometric analysis, proline-179 (FIGS.3A-C) appears to be the C-terminal amino acid in the major soluble formfound in A498 cell-conditioned media. An alternate form with alanine-177(FIGS. 3A-C) at the C-terminus was also detected.

B. Expression of Recombinant HEK4 BP

The HEK4 BP cDNA clone shown in FIGS. 3 was inserted into the plasmidvector pDSRα for expression mammalian cells. The recombinant plasmid wastransfected into Chinese hamster ovary (CHO) cells by calcium phosphateprecipitation and cells containing the plasmid were selected by growthin DMEM (high glucose, GIBCO, Bethesda, MD), 1×penicillin/streptomycin/glutamine (PSG), 1× non-essential amino acids(NEAA) containing 10% fetal bovine serum (FBS), but lacking HTsupplement (HT supplement: 10 mM sodium hypoxanthine, 1.6 mM thymidine).The expression of HEK4 BP in several clones was assessed by the level ofHEK4-receptor binding activity in each clone's cell-conditioned media asdetermined by BIAcore (Pharmacia Biosensor, Piscataway, N.J.). Thiscorrelated well with the level of HEK4 BP mRNA in the clones asdetermined by Northern blot hybridization. One clone, CHO/HL6, was asignificantly better producer of recombinant HEK4 BP than the others andwas chosen for further work. Expression of HEK4 BP by CHO/HL6 wasenhanced 2 to 4-fold by treatment with increasing amounts ofmethotrexate up to 100 nM over a period of several weeks. Followingamplification, cells expressing HEK4 BP were expanded and transferred toroller bottles for production of conditioned media to be used as asource for purification of the recombinant protein. A total of 100roller bottles were seeded with CHO/HL6 cells at 10⁷ cells per bottle.Cells were grown until confluent (approximately 4 days) in DMEM (highglucose, GIBCO, Bethesda, Md.) , 1× PSG, 1× NEAA, and 10% FBS. Followingthe growth phase, the media was removed replaced with the same media butwith 0.5% rather than 10% FBS. After 3 days, the media was collected,filtered to remove any cell debris, and stored frozen at -80° C.

EXAMPLE 4 Purification of Recombinant HEK4 BP

Approximately 20-25 liters of conditioned medium from CHO/HL6 cells werethawed at room temperature and filtered. The medium was concentrated anddiafiltered against 10 mM Tris-HCl, pH 8.5 (4° C.) using a 10,000molecular weight cut off membrane. The diafiltrate was applied to acolumn of Q-Sepharose, High Performance and subsequently eluted with alinear gradient of NaCl (0-0.3 M) in 10 mM Tris-HCl, pH 8.5. Fractionswere analyzed for the presence of HEK4 BP by immunoblotting using anantibody generated against unfolded HEK4 BP produced in E. coli, or bybinding to HEK4 receptor immobilized on a BIAcore sensor chip. Fractionscontaining HEK4 BP were pooled, concentrated and applied to a gelfiltration column. (Superdex 75, 5×85 cm, PBS, 3 mL/min). Fractionscontaining HEK4 BP were further purified by C4 reverse phase HPLC (Vydac214TP 4.6×250 mm, 2.9 mL/min) using an acetonitrile gradient (22-44%) in0.1% trifluoroacetic acid. The column profile and SDS-PAGE analysis ofpeak fractions are shown in FIGS. 4A, 4B. Fractions were evaporatedunder vacuum and formulated in 0.25 M Tris-HCl, 2 mM CHAPS, pH 7.5.

EXAMPLE 5 A. Production of Antibodies to HEK4 Receptor

Antibodies directed against the HEK4 receptor extracellular domain wereproduced using standard methods (Harlow and Lane, Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988)). cDNA encoding amino acids 22-148 of the HEK4receptor was inserted into the pATH vector (Yansura, 1990) in the sametranslational reading frame as the TrpE gene. The resulting plasmid wasintroduced into an E. coli host strain resulting in expression of theHEK4/TrpE fusion protein. Bacterial cell lysates were fractionated bypreparative SDS-PAGE and the band containing the HEK4/TrpE fusion wasexcised. The crushed gel slice was used to immunize rabbits according tostandard protocols (Harlow and Lane, supra). The antisera generated bythis method recognized both the HEK4/TrpE antigen and recombinant HEK4-Xproduced in CHO cells.

Antibodies to the C-terminal 12 amino acids of the HEK4 receptor(sequence is cys-leu-glu-thr-gln-ser-lys-asn-gly-pro-val-pro-val) wereproduced by the same method (Harlow and Lane, supra) using a syntheticpeptide chemically linked to keyhole limpet hemocyanin (KLH) as theantigen. The antiserum was purified by passage over a column upon whichpeptide antigen had been immobilized using a SulfoLink kit (Pierce,Rockford, Ill.). These antibodies were able to specifically recognizeHEK4 receptor by Western blots.

B. Production of Antibodies to HEK4 BP

HEK4 BP cDNA as shown in FIG. 3 was used as a template for PCR withprimers 819-31 and 819-28 to produce a polypeptide fragment coding foramino acids 1-179 of HEK4 BP (FIGS. 3A, 3B, 3C).

819-31) 5' GGAGGACATATGAGCCAGGACCCGGGCTCCAAG 3' (SEQ ID NO:10)

819-28) 5' GAAGAAGGATCCCTATGGCTCGGCTGACTCATGTAC 3'

(SEQ ID NO:11)

The PCR fragment was cloned into the expression vector pCFM1656 usingthe NdeI and BamHI sites included in the primers. The resultingrecombinant plasmid was transformed into E. coli FM5 (ATCC No. 53911)and the truncated HEK4 BP was expressed as insoluble inclusion bodies.The inclusion bodies were solubilized and the HEK4 BP fragment purifiedby SDS-polyacrylamide gel electrophoresis was used as an antigen inrabbits. The antisera was generated and characterized as described(Harlow and Lane, supra) and recognized HEK4 BP expressed in CHO cellsby Western blot analysis.

EXAMPLE 6 A. Expression Pattern of the HEK4 Receptor

The expression of HEK4 receptor mRNA in rat and human tissues has beenpreviously reported in co-pending and commonly owned U.S. Ser. No.08/229,509, relevant portions of which have been incorporated herein byreference. The results of these studies are summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                        Tissue Distribution of HEK4 Receptor                                                 Tissue:      Human        Rat                                          ______________________________________                                        Brain           ++           +                                                  Heart + bd                                                                    Kidney + bd                                                                   Liver + bd                                                                    Lung + +                                                                      Muscle + bd                                                                   Ovary nt bd                                                                   Pancreas + nt                                                                 Placenta +++ nt                                                               Stomach nt bd                                                                 Testis nt +                                                                   Thymus nt bd                                                                ______________________________________                                         bd = below detection                                                          nt = not tested                                                          

In the human tissues studied, HEK4 receptor mRNA is most abundantlyexpressed in placenta, with lower levels in heart, brain, lung, andliver. Previous studies on HEK4 receptor mRNA in cell lines foundexpression in one pre-B cell line and two T-cell lines (Wicks et al.1992).

B. Expression Pattern of HEK4 BP

The expression of HEK4 BP mRNA in human tissues was examined by Northernblot analysis. A Northern blot containing 2 μg of polyA⁺ from each ofthe tissues indicated was purchased from Clontech (Palo Alto, Calif.)and hybridized with a ³² P-labeled HEK4 BP probe. As shown in FIG. 5,HEK4 BP mRNA is expressed at high levels in human adult brain, kidney,and placenta. Readily detectable levels can also be found in heart,lung, liver, spleen, prostate, testis, ovary, small intestine, andcolon. The presence of HEK4 BP mRNA in many different tissues isconsistent with the idea that this factor is important for thedifferentiation, development, and/or maintenance of a variety of celltypes.

EXAMPLE 7 A. HEK4 BP Activation of EPH Subfamily Receptors by Cell-CellAutophosphorylation

The hallmark of receptor activation for all known receptorprotein-tyrosine kinases is autophosphorylation (van der Geer et al.,supra). To determine whether HEK4 BP can activate HEK4 receptor, acell-cell autophosphorylation assay was performed. Recipient cells wereCHO cells transfected with HEK4 receptor cDNA which had beenserum-starved by incubation in media with 0.5% serum for 16 hours. Thedonor cells were CHO cells transfected with HEK4 BP cDNA (see Example3B) or CHO cells that had been transfected with vector alone. Donorcells were scraped from the surface of their growth vessel inphosphate-buffered saline and added to recipient cells for 30 minutes at37° C. After washing, the recipient cells were lysed in modified RIPAbuffer (10 mM sodium phosphate, pH 7.4, 150 mM sodium chloride, 0.1%sodium dodecyl sulfate, 1% NP-40, 1% deoxycholate, 10 mg/ml aprotinin, 5mM EDTA, 200 mM sodium orthovanadate). Receptors were immunoprecipitatedfrom the cell lysate and prepared for SDS polyacrylamide gelelectrophoresis as previously described (Bartley et al., supra). Afterelectrophoresis and electroblotting to membranes, the immunoprecipitateswere probed with antiphosphotyrosine antibodies (4G10, UBI, Lake Placid,N.Y.). Immune complexes were detected by horseradish peroxidaseconjugated secondary reagents using chemiluminescence as described bythe manufacturer (ECL, Amersham). As shown in FIGS. 6A, 6B cellsexpressing HEK4 binding protein were able to stimulate tyrosinephosphorylation on both the HEK and ECK receptors. Control cells did notstimulate the phosphorylation of either receptor. The resultsdemonstrate that HEK4 BP can activate both the HEK4 and the ECKreceptors.

B. Activation of HEK4 Receptor by Soluble HEK4 BP

To determine whether soluble recombinant HEK4 BP could activate the HEK4receptor, CHO cells transfected with HEK4 receptor cDNA were treatedwith conditioned media from CHO cells expressing HEK4 BP (see Example3B) or with purified recombinant HEK4 BP (see Example 4). The cells wereserum-starved by incubation in media with 0.5% serum for 16 hours priorto the treatments. Treatments were for 30 minutes at 37° C., after whichthe cells were lysed in modified NP40 buffer (50 mM Tris, pH 8.0, 150 mMsodium chloride, 1% NP40, 10 mg/ml aprotinin, 5 mM EDTA, 200 mM sodiumorthovanadate), HEK4 receptor was immunoprecipitated, and prepared forSDS polyacrylamide gel electrophoresis as previously described (Bartleyet al., supra). After electrophoresis and electroblotting to membranes,the immunoprecipitates were probed with antiphosphotyrosine antibodies(4G10, UBI, Lake Placid, N.Y.). Immune complexes were detected byhorseradish peroxidase conjugated secondary reagents usingchemiluminescence as described by the manufacturer (ECL, Amersham). Asshown in FIGS. 7A, 7B soluble recombinant HEK4 BP in conditioned mediaand after purification activated HEK4 receptor. This activation wasenhanced by pretreatment of conditioned media or purified HEK4 BP withthe antibodies of Example 5B which had been affinity purified on a HEK4BP column. Antibodies (-50 μg/ml) were incubated with conditioned mediumor purified HEK4 BP at 4° for 1 hr. prior to treatment of CHO cellsexpressing HEK4 receptor.

EXAMPLE 8 Affinity of HEK4 BP for EPH-like Receptors

A competition assay was used to measure differences in HEK4 BP bindingto different EPH-like receptors. Purified HEK4 BP was incubated withvarious concentrations of either HEK4, HEK8 or ECK soluble receptors andbinding of the mixture to immobilized HEK4 receptor was analyzed byBIAcore. The concentration of soluble receptor that inhibited HEK4 BPbinding by 50% is termed IC50. IC50 values allow a comparison of therelative affinity of HEK4 BP for related receptors.

IC50 values were determined as follows. Analysis of HEK4 BP binding toimmobilized HEK4 receptor showed a linear response in the range 60 to500 ng/ml. Various amounts of the purified extracellular domains ofHEK4, ECK, or HEK8 were incubated with 0.250 μg/ml HEK4 BP prepared asdescribed in Example 4 in solutions containing 100 μg/ml BSA, 10 mMHEPES, 0.15M NaCl, 3.4 mM EDTA, and 1 mg/ml soluble dextran, pH 7.4.These solutions were incubated for at least 30 minutes at 3° C. prior toinjection. Protein concentrations of receptor stocks were confirmed byBCA protein assay. Duplicates of each sample were run in parallel withstandard curves on two different days. All surfaces were regenerated towithin 10 RU of baseline with 25 mM CAPS and 1 M NaCl pH 10.4. The meanbinding response was plotted versus soluble receptor concentration(FIGS. 8) providing IC50 values of 0.55 ug/ml for HEK4, 5.0 μg/ml forECK, and 10.5 μg/ml for HEK8. Thus, HEK4 BP preferentially binds HEK4receptor compared to two other EPH family members, ECK and HEK8.

While the invention has been described in what is considered to be itspreferred embodiments, it is not to be limited to the disclosedembodiments, but on the contrary, is intended to cover variousmodifications and equivalents included within the spirit and scope ofthe appended claims, which scope is to be accorded the broadestinterpretation so as to encompass all such modifications andequivalents.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 11                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1728 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 175..858                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 232..858                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 175..231                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GAATTCCCCC AGCTTGGTGG GCGCCTCTTT CCTTTCTCGC CCCCTTTCAT TT -            #TTATTTAT     60                                                                 - - TCATATTTAT TTGGCGCCCG CTCTCTCTCT GTCCCTTTGC CTGCCTCCCT CC -            #CTCCGGAT    120                                                                 - - CCCCGCTCTC TCCCCGGAGT GGCGCGTCGG GGGCTCCGCC GCTGGCCAGG CG - #TG        ATG      177                                                                                      - #                  - #                  - #             Met                                                                                               - #                  - #                  - #             -19                                                                              - - TTG CAC GTG GAG ATG TTG ACG CTG GTG TTT CT - #G GTG CTC TGG ATG        TGT      225                                                                    Leu His Val Glu Met Leu Thr Leu Val Phe Le - #u Val Leu Trp Met Cys                      -15      - #           -10      - #            -5                  - - GTG TTC AGC CAG GAC CCG GGC TCC AAG GCC GT - #C GCC GAC CGC TAC GCT          273                                                                       Val Phe Ser Gln Asp Pro Gly Ser Lys Ala Va - #l Ala Asp Arg Tyr Ala                     1        - #       5           - #       10                          - - GTC TAC TGG AAC AGC AGC AAC CCC AGA TTC CA - #G AGG GGT GAC TAC CAT          321                                                                       Val Tyr Trp Asn Ser Ser Asn Pro Arg Phe Gl - #n Arg Gly Asp Tyr His            15                 - # 20                 - # 25                 - # 30       - - ATT GAT GTC TGT ATC AAT GAC TAC CTG GAT GT - #T TTC TGC CCT CAC TAT          369                                                                       Ile Asp Val Cys Ile Asn Asp Tyr Leu Asp Va - #l Phe Cys Pro His Tyr                            35 - #                 40 - #                 45              - - GAG GAC TCC GTC CCA GAA GAT AAG ACT GAG CG - #C TAT GTC CTC TAC ATG          417                                                                       Glu Asp Ser Val Pro Glu Asp Lys Thr Glu Ar - #g Tyr Val Leu Tyr Met                        50     - #             55     - #             60                  - - GTG AAC TTT GAT GGC TAC AGT GCC TGC GAC CA - #C ACT TCC AAA GGG TTC          465                                                                       Val Asn Phe Asp Gly Tyr Ser Ala Cys Asp Hi - #s Thr Ser Lys Gly Phe                    65         - #         70         - #         75                      - - AAG AGA TGG GAA TGT AAC CGG CCT CAC TCT CC - #A AAT GGA CCG CTG AAG          513                                                                       Lys Arg Trp Glu Cys Asn Arg Pro His Ser Pr - #o Asn Gly Pro Leu Lys                80             - #     85             - #     90                          - - TTC TCT GAA AAA TTC CAG CTC TTC ACT CCC TT - #T TCT CTA GGA TTT GAA          561                                                                       Phe Ser Glu Lys Phe Gln Leu Phe Thr Pro Ph - #e Ser Leu Gly Phe Glu            95                 - #100                 - #105                 - #110       - - TTC AGG CCA GGC CGA GAA TAT TTC TAC ATC TC - #C TCT GCA ATC CCA GAT          609                                                                       Phe Arg Pro Gly Arg Glu Tyr Phe Tyr Ile Se - #r Ser Ala Ile Pro Asp                           115  - #               120  - #               125              - - AAT GGA AGA AGG TCC TGT CTA AAG CTC AAA GT - #C TTT GTG AGA CCA ACA          657                                                                       Asn Gly Arg Arg Ser Cys Leu Lys Leu Lys Va - #l Phe Val Arg Pro Thr                       130      - #           135      - #           140                  - - AAT AGC TGT ATG AAA ACT ATA GGT GTT CAT GA - #T CGT GTT TTC GAT GTT          705                                                                       Asn Ser Cys Met Lys Thr Ile Gly Val His As - #p Arg Val Phe Asp Val                   145          - #       150          - #       155                      - - AAC GAC AAA GTA GAA AAT TCA TTA GAA CCA GC - #A GAT GAC ACC GTA CAT          753                                                                       Asn Asp Lys Val Glu Asn Ser Leu Glu Pro Al - #a Asp Asp Thr Val His               160              - #   165              - #   170                          - - GAG TCA GCC GAG CCA TCC CGC GGC GAG AAC GC - #G GCA CAA ACA CCA AGG          801                                                                       Glu Ser Ala Glu Pro Ser Arg Gly Glu Asn Al - #a Ala Gln Thr Pro Arg           175                 1 - #80                 1 - #85                 1 -      #90                                                                              - - ATA CCC AGC CGC CTT TTG GCA ATC CTA CTG TT - #C CTC CTG GCG ATG        CTT      849                                                                    Ile Pro Ser Arg Leu Leu Ala Ile Leu Leu Ph - #e Leu Leu Ala Met Leu                          195  - #               200  - #               205              - - TTG ACA TTA TAGCACAGTC TCCTCCCATC ACTTGTCACA GAAAACATC - #A                  898                                                                       Leu Thr Leu                                                                    - - GGGTCTTGGA ACACCAGAGA TCCACCTAAC TGCTCATCCT AAGAAGGGAC TT -             #GTTATTGG    958                                                                 - - GTTTTGGCAG ATGTCAGATT TTTGTTTTCT TTCTTTCAGC CTGAATTCTA AG -            #CAACAACT   1018                                                                 - - TCAGGTTGGG GGCCTAAACT TGTTCCTGCC TCCCTCACCC CACCCCGCCC CA -            #CCCCCAGC   1078                                                                 - - CCTGGCCCTT GGCTTCTCTC ACCCCTCCCA AATTAAATGG ACTCCAGATG AA -            #AATGCCAA   1138                                                                 - - ATTGTCATAG TGACACCAGT GGTTCGTCAG CTCCTGTGCA TTCTCCTCTA AG -            #AACTCACC   1198                                                                 - - TCCGTTAGCG CACTGTGTCA GCGGGCTATG GACAAGGAAG AATAGTGGCA GA -            #TGCAGCCA   1258                                                                 - - GCGCTGGCTA GGGCTGGGAG GGTTTTGCTC TCCTATGCAA TATTTATGCC TT -            #CTCATTCA   1318                                                                 - - GAACTGTAAG ATGATCGCGC AGGGCATCAT GTCACCATGT CAGGTCCGGA GG -            #GGAGGGCC   1378                                                                 - - TATCCCCCTA TCCCAGGCAT CCCAGACGAG GACTGGCTGA GGCTAGGCGC TC -            #TCATGATC   1438                                                                 - - CACCTGCCCC GGGAGGGCAG CGGGGAAGAC AGAGAAAAGC AAAACGCATT CC -            #TCCTCAGC   1498                                                                 - - TCCACCCACC TGGAGACGAA TGTAGCCAGA GAGGAGGAAG GAGGGAAACT GA -            #AGACACCG   1558                                                                 - - TGGCCCCTCG GCCTTCTCTC TGCTAGAGTT GCCGCTCAGA GGCTTCAGCC TG -            #ACTTCCAG   1618                                                                 - - CGGTCCCAAG AACACCTACT AATTCTTCTC CACTCCTTCA TGGCTGGGAC AG -            #TTACTGGT   1678                                                                 - - TCATATGCAA GTAAAGATGA CAATTTACTC AACAAAAAAA AAAGGAATTC  - #                1728                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 228 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Leu His Val Glu Met Leu Thr Leu Val Ph - #e Leu Val Leu Trp Met     19             -15    - #             -10    - #              -5                - - Cys Val Phe Ser Gln Asp Pro Gly Ser Lys Al - #a Val Ala Asp Arg Tyr                    1    - #           5       - #           10                      - - Ala Val Tyr Trp Asn Ser Ser Asn Pro Arg Ph - #e Gln Arg Gly Asp Tyr           15             - #     20             - #     25                          - - His Ile Asp Val Cys Ile Asn Asp Tyr Leu As - #p Val Phe Cys Pro His       30                 - # 35                 - # 40                 - # 45       - - Tyr Glu Asp Ser Val Pro Glu Asp Lys Thr Gl - #u Arg Tyr Val Leu Tyr                       50 - #                 55 - #                 60              - - Met Val Asn Phe Asp Gly Tyr Ser Ala Cys As - #p His Thr Ser Lys Gly                   65     - #             70     - #             75                  - - Phe Lys Arg Trp Glu Cys Asn Arg Pro His Se - #r Pro Asn Gly Pro Leu               80         - #         85         - #         90                      - - Lys Phe Ser Glu Lys Phe Gln Leu Phe Thr Pr - #o Phe Ser Leu Gly Phe           95             - #    100             - #    105                          - - Glu Phe Arg Pro Gly Arg Glu Tyr Phe Tyr Il - #e Ser Ser Ala Ile Pro      110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - Asp Asn Gly Arg Arg Ser Cys Leu Lys Leu Ly - #s Val Phe Val Arg        Pro                                                                                             130  - #               135  - #               140             - - Thr Asn Ser Cys Met Lys Thr Ile Gly Val Hi - #s Asp Arg Val Phe Asp                  145      - #           150      - #           155                  - - Val Asn Asp Lys Val Glu Asn Ser Leu Glu Pr - #o Ala Asp Asp Thr Val              160          - #       165          - #       170                      - - His Glu Ser Ala Glu Pro Ser Arg Gly Glu As - #n Ala Ala Gln Thr Pro          175              - #   180              - #   185                          - - Arg Ile Pro Ser Arg Leu Leu Ala Ile Leu Le - #u Phe Leu Leu Ala Met      190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - Leu Leu Thr Leu                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGATCTAGAG CACCAGCAAC ATGGATTGT         - #                  - #                29                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TCGGTCTAGA TCATTATTGG CTACTTTCAC CAGAGAT      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Val Asn Phe Asp Gly Tyr Ser Ala Arg Asp                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Val Phe Asp Val Asn Phe Lys Val Glu Xaa Se - #r Leu Glu Pro Ala        Asp                                                                             1               5   - #                10  - #                15              - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ala Val Ala Asp Arg Tyr Ala Val Tyr Trp As - #n Ser Ser Asn Pro Arg      1               5   - #                10  - #                15               - - Phe Gln Arg Gly Asp Tyr His Ile Ile Val Xa - #a Ile Asn Xaa Tyr                      20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GAYMGNTAYG CNGTNTAYTG G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - RTANCCRTCR AARTTNACCA T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGAGGACATA TGAGCCAGGA CCCGGGCTCC AAG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GAAGAAGGAT CCCTATGGCT CGGCTGACTC ATGTAC      - #                  -     #       36                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid encoding a polypeptidewhich binds to at least a HEK4 receptor wherein the nucleic acid isselected from the group consisting of:a) the nucleic acid having thesequence shown in SEQ ID NO:1 and its complementary strand; b) nucleicacids which hybridize under conditions of 6×SSC and 65° C. and remainhybridized under conditions of 0.2×SSC and 65° C. with the coding regionof SEQ ID NO: 1; and c) nucleic acids which are degenerate to nucleicacids set forth in (a) and (b).
 2. The nucleic acid of claim 1 which iscDNA, genomic DNA or synthetic DNA.
 3. The nucleic acid of claim 1 whichcomprises one or more codons preferred for expression in Escherichiacoli.
 4. An expression vector comprising the nucleic acid of claim
 1. 5.A procaryotic or eucaryotic host cell stably transformed or transfectedwith the vector of claim
 4. 6. The host cell of claim 5 which is a CHOcell.
 7. The host cell of claim 5 which is Escherichia coli.
 8. Aprocess for the production of a polypeptide which binds at least a HEK4receptor comprising growing under suitable nutrient conditions aprocaryotic or eukaryotic host cell transformed or transfected with thenucleic acid of claim 1 and isolating the polypeptide product of theexpression of the nucleic acid.